Dec 23, 2024
Molecular Dynamics Simulations of Active, Membrane-Engaged Complex
Forked from Molecular Dynamics Simulations
- Annan SI Cook1,2,
- Shanlin Rao2,3,
- Ainara Claveras Cabezudo2,4,3
- 1University of California, Berkeley;
- 2Aligning Science Across Parkinson's CRN;
- 3Max Planck Institute of Biophysics;
- 4IMPRS on Cellular Biophysics
Protocol Citation: Annan SI Cook, Shanlin Rao, Ainara Claveras Cabezudo 2024. Molecular Dynamics Simulations of Active, Membrane-Engaged Complex. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm9px5l3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 19, 2024
Last Modified: December 23, 2024
Protocol Integer ID: 116412
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000350
Abstract
This protocol details molecular dynamics simulations of the active PI3KC3-C1 complex bound to RAB1A on a lipid membrane.
Attachments
852-2196.pdf
38KB
Materials
Material
aqueous NaCl
Model Preparation
Model Preparation
Prepare all-atom structural models of PI3KC3-C1 based on the active conformation with missing regions predicted by AlphaFold-Multimer 2.2.
Assign protonation states to amino acid side chains based on pKa predictions by PROPKA.
Parameterize ATP
Parameterize ATP
Parameterize a molecule of ATP in the VPS34 active site using CHARMM-GUI.
Membrane Construction
Membrane Construction
Construct lipid membranes consisting of DOPC (60%), DOPE (20%), DOPS (5%), and POPI (15%) using the insane method.
Randomly distribute coarse-grained lipids in the membrane.
Solvation and Equilibration
Solvation and Equilibration
Solvate each membrane patch with 150 millimolar (mM) aqueous NaCl.
Perform an initial equilibration of 200 ns.
Conversion to Atomistic Representation
Conversion to Atomistic Representation
Convert the equilibrated membrane patch into an atomistic representation using the CG2AT2 tool.
Protein-Membrane System Setup
Protein-Membrane System Setup
Place atomistic PI3KC3-C1 above the resulting membranes with a minimum distance of ~2 nm between protein and lipid atoms.
Further Equilibration
Further Equilibration
Subject the protein-membrane systems to an additional 10 ns of equilibration.
Apply harmonic positional restraints (force constant: 1000 kJ mol⁻¹ nm⁻²) to non-hydrogen protein atoms during this equilibration phase.
Production Runs
Production Runs
Perform 2 µs production runs for each of the six independent replicates of the simulation system.
Maintain system pressure at 1 Bar and temperature at 310 °К using the Parrinello-Rahman barostat and velocity-rescaling thermostat.
Electrostatic Interactions
Electrostatic Interactions
Treat long-range electrostatic interactions using the smooth particle mesh Ewald method with charge interpolation through fourth-order B-splines.
Integration Time Step
Integration Time Step
Use a 2 fs integration time step for the simulations.
Bond Constraints
Bond Constraints
Apply the LINCS algorithm to constrain covalent bonds involving hydrogen atoms.
Steered Molecular Dynamics (SMD)
Steered Molecular Dynamics (SMD)
For SMD simulations, apply harmonic restraints (force constant: 100 kJ mol⁻¹) to reduce the center-of-mass z-distance between the protein group of interest and the membrane lipids underneath.
Apply a negative rate of -0.5 nm ns⁻¹ until the protein group reaches the membrane surface.
Relaxation
Relaxation
Allow the complex to relax over periods of 2 µs during further simulations upon removal of any steering force.
Preparation of membrane-engaged complex containing RAB1A
Preparation of membrane-engaged complex containing RAB1A
Superimpose the cryo-EM structure of the active-state complex including RAB1A onto the final structure of the previous MD run.
Model the two RAB1A geranylgeranyl groups and the VPS15 N-myristate using CHARMM-GUI.
Repeat above-described SMD protocol to adjust the remaining membrane interactions.
Allow the complex to relax for 1 µs (3 independent replicas with random velocities).